The present invention relates to the identification of Toxoplasma gondii antigens and the preparation thereof by genetic engineering. A cDNA expression gene bank of this parasite was prepared. Recombinant clones which are of diagnostic interest were identified using a high-titer rabbit anti-Toxoplasma gondii serum, and isolated.
Toxoplasma gondii (T.gondii) is an obligatory intra-cellular single-cell parasite which is categorized as a coccidium. The parasite has a relatively wide range of hosts and can, in addition to very many mammals, also infect man. In the latter case there are two forms which differ from each other physiologically: xe2x80x9ctachyzoitesxe2x80x9d reproduce asexually in a number of different cell types. This form is found exclusively in the acute stage of the infection. xe2x80x9cBradyzoitesxe2x80x9d, in contrast, persist in cells of the cardiac and skeletal muscles and in cells of the central nervous system in encapsulated form and are responsible for a persistent immunity to reinfection. It is estimated that globally there are 500 million people who are chronically infected by T.gondii. 
In healthy adults, a T.gondii infection normally has no symptoms with the exception of a slight swelling of the lymph nodes. During pregnancy and in immunosuppressed patients, however, an infection with this parasite may present particular problems. Thus there is the risk of an intra-uterine transfer of these parasites in pregnant women who have not acquired a protection from T.gondii by immunity. This leads to the infection of the fetus and may result in deformities of the child or the expulsion of the fetus.
Immunosuppressed patients frequently acquire an acute T.gondii infection as a result of the reactivation of enzysted xe2x80x9cbradyzoitesxe2x80x9d. In most cases this leads to cerebral toxoplasmosis (encephalitis), which may, under certain circumstances, be lethal. In addition to cerebral toxoplasmosis, T.gondii has also been mentioned as causative agent of eye diseases (chorioretinitis). These cases, too, are infections which can be blamed on a reactivation of xe2x80x9cbradyzoitesxe2x80x9d.
The clinical picture of toxoplasmosis often causes difficulties concerning differential diagnosis to the clinician so that the support by laboratory analyses in establishing the diagnosis is sought. The detection of antibodies and the determination of the titer or of the dynamics of the titer have therefore become essential tools for diagnosing toxoplasmosis. Methods for determining toxoplasma-specific immounoglobulins of the G and M class, such as indirect immunofluorescence (IF), complement fixation reaction (CF), indirect hemagglutination (IHA), latex agglutination (LA) and enzyme-linked immuno-assay (ELISA) are very common in the field of serodiagnosis but often have faults. For example these test methods vary very greatly as regards specificity and sensitivity. These differences are primarily caused by the preparation of the antigen which is used for the serological test. In most cases total cell antigen which contains a high proportion of unspecific cell components and is held responsible for the occurrence of false positive test results, is prepared. In addition, obtaining the antigens from infected mice holds the risk of infection for the person working in the laboratory.
In view of the specificity and sensitivity of a diagnostic of this type, it would thus be desirable to employ defined immunoreactive antigens which should additionally make it possible to distinguish between IgG- and IgM-specific anti-T.gondii antibodies.
A number of antigens of diagnostic interest have been described for T.gondii in the literature. For example Hughes describes in a review (Curr. Top. Microbiol. (1985), 120: 105-139) four major antigens which are potentially suitable for detecting anti-T.gondii antibodies of the IgG class, having molecular weights of 45, 32, 27 and 21 kilodalton (kD). Handman et al. (Immunol. (1980), 40: 579-588) and Potasman et al. (J. Infect. Diseases (1986), 154: 650-657) analyzed sera taken throughout the course of the disease of acutely infected T.gondii patients using Western blots and demonstrated that a 35 kD membrane antigen reacts with IgG antibody at a very early stage. Decoster et al. (Clinic. Exper. Immunol. (1988), 73: 376-382) describe four antigens of diagnostic interest, which, in contrast to the 35 kD antigen, can be isolated from the culture medium and have been termed xe2x80x9cexcreted-secreted antigensxe2x80x9d (ES antigens) and which have molecular weights of 105, 97, 66 and 28.5 kD. IgG antibodies which react with antigens of 105, 97 and 28.5 kD seem to be good markers for a chronic toxoplasmosis. Similarly to the 35 kD antigen, the 97 kD antigen and the 66 kD antigen are recognized at a very early stage by IgM antibodies of acutely infected patients. It has to be pointed out that these antigens have not been sufficiently characterized by giving a molecular weight after electrophoretic fractionation because there usually are several proteins within one molecular weight range.
A 6 kD antigen is a further marker for acute toxoplasmosis (Ehrlich et al., (1983), Infect. Immun. 41: 683-690). In IgM Western blots, this antigen reacts relatively strongly. To date there are only very few data which might reveal the nature of this antigen.
Only very few T.gondii antigens have been biochemically characterized so far. The main surface protein P30 is an exception. This antigen is a glycoprotein which is anchored in the membrane via a glycolipid (Nagel et al., (1989), J. Biol. Chem. 264: 5569-5576). The diagnostic importance of this antigen is controversial since P30 also reacts with unspecific antibodies of the IgG class (Potasman et al., (1986), J. Clin. Microbiol. 24: 1050-1054).
The isolation and purification of individual antigens for the use in serodiagnosis often involves a considerable amount of work. Both the molecular weight data and the classification of the immunoreactivity of an antigen can substantially differ from case to case in conventionally purified antigen. Cloning and expressing such antigens and investigating the structure of the corresponding genes might not only improve the yield of purified antigen but should also contribute to the serological characterization and therefore to the investigation of the diagnostic relevance of the antigen. So far the structure of the genes of two immunologically interesting T.gondii antigens has been investigated. The complete nucleotide sequences of these antigens, which are P30 (Burg et al., (1988), J. Immunol. 141: 3584-3591) and a 28 kD antigen (Prince et al., (1989), Mol. Biochem. Parasitol. 34: 3-14), are known.
The object of the present invention is to prepare by genetic engineering defined antigens of T.gondii, which are suitable for diagnosis and prevention. It has been possible to successfully identify suitable T.gondii gene products from a lambda gt11 cDNA expression gene bank using a high-titer rabbit anti-T.gondii serum. Partial nucleic acid sequences, and aminoacid sequences derived therefrom, of 8 clones (F2, F28, F29, F34, F45, F61, F74 and F76) have been determined. All the abovementioned clones react in Western blots with human anti-T.gondii IgG sera. The clones F34, F61 and F76 additionally react with specific antibodies of the IgM class. The partial nucleotide sequences are listed in Tab. 1-8 (SEQ ID NOS:1-14) and, as far as they are apparent, also the translational reading frames (in Tab. 1-6 corresponding to SEQ ID NOS:1-12).
F61 (Tab. 1 SEQ ID NOS:1-2) is assigned to a protein having a molecular weight of 66 kD.
F34 (Tab. 2 SEQ ID NOS:3-4) belongs to a protein of about 68 kD.
F29 (Tab. 3 SEQ ID NOS:5-6) belongs to a protein of about 30 kD.
F28 (Tab. 4 SEQ ID NOS:7-8) belongs to a protein of about 28 kD.
F2 (Tab. 5 SEQ ID NOS:9-10) belongs to a protein of about 30 kD.
F76 (Tab. 6 SEQ ID NOS:11-12)belongs to a protein of about 35 kD.
F45 (Tab. 7 SEQ ID NOS:13) belongs to a protein of about 29 kD.
F74 (Tab. 8 SEQ ID NOS:14) belongs to a protein of about 64 kD.
With the aid of the partial sequences mentioned it is readily possible to clone the complete genes for the abovementioned partial sequences.
The partial sequences depicted in the Tables 1, 2 and 6 (SEQ ID NOS:1-12) were accordingly used to complete the coding cDNA regions of the genes belonging thereto. For this purpose, the cDNAs F61, F34 and F76 were radiolabeled and used as probes for screening the cDNA gene bank. The sequence from Table 1, F61, was used to isolate the cDNA of the P66 protein. The sequence from Tab. 2 (SEQ ID NOS:3-4), F34, was used for the isolation of the cDNA of the P68 protein. For the isolation of the cDNA of the P35 protein, the sequence from Tab. 6 (SEQ ID NOS:11-12), F76, was used. Recombinant clones having homologies to these sequences were isolated and characterized structurally by sequencing the inserted T.gondii-specific cDNA regions. The nucleotide sequences of the complete ranges of the structural genes of the P35, P66 and P68 proteins are depicted in the Tables 9-11 (SEQ ID NOS:15-20).
Immunologically reactive partial regions (immunogenic parts) are representatively described for P35, P66 and P68 in the Examples 6 and 7. Other immunogenic protein regions are tested or determined in an analogous way. The invention therefore relates to
(a) the isolated inserted DNA sequences of the abovementioned clones, including the transcription products thereof and the remaining sequences to complete the particular structural genes,
(b) DNA structures and vectors which contain, completely or in part, these sequences,
(c) prokaryotic or eukaryotic cells which have been transformed with DNA of this type,
(d) the polypeptides expressed by transformed cells of this type, or immunogenic parts thereof including the use thereof for diagnosis and therapy or prevention,
(e) the amino-acid sequences (AS) belonging thereto,
(f) antibodies against the polypeptides under (d), including the use thereof for the diagnosis and therapy or prevention of T.gondii infections, and
(g) processes for the preparation by genetic engineering of the polypeptides mentioned under (d) or of immunogenic parts thereof.
The invention is furthermore described in the examples and the claims.